JP7417294B2 - Cross flow filtration device - Google Patents

Description

The present invention relates to a cross-flow filtration device suitable for separating and purifying microparticles such as cultured cells, blood components, organelles, microorganisms, various synthetic microparticles, environmental microparticles, liposomes, and vesicles.

Various synthetic particles made of polymers and inorganic materials, as well as fine particles such as liposomes and vesicles, are widely used in fields such as precision mechanical engineering, functional materials, separation processes, and drug delivery. In preparing various types of synthetic fine particles, such as separation carriers for chromatography, liquid crystal spacers, fine particles for electronic paper, and carriers for drug delivery, it is essential to make the size uniform. Therefore, a technique for efficiently separating and purifying specific fine particles present in a mixture of fine particles is essential.

For example, various diseases can be diagnosed by separating and purifying specific cells and biological microparticles present in the blood and analyzing the functions and abundance ratios of these microparticles. For example, by quantitatively evaluating circulating cancer cells present in the blood, it becomes possible to diagnose cancer early and verify the efficacy of cancer treatments. Furthermore, by isolating and analyzing fetal-derived nucleated red blood cells present in a pregnant woman's blood, a definitive diagnosis of chromosomal abnormalities can be made. Furthermore, in recent years, it is expected that it will become possible to diagnose various diseases by analyzing microscopic vesicles and exosomes with a diameter of about 50 to 1000 nanometers that exist in the blood.

For example, in the differentiation of stem cells such as iPS cells, selecting only cells in a specific differentiation state is essential in regenerative medicine and drug evaluation model development. In addition, monocytes and various mesenchymal stem cells present in the blood are useful in nerve regeneration medicine, cancer immunotherapy, peripheral blood vessel regeneration, etc., so these specific cells can be efficiently harvested. Separation and purification technology is important.

Various methods have been proposed so far as techniques for separating and selecting particles having specific properties (for example, size) from a complex population of particles. Typical examples include centrifugation, sedimentation, filtration, and separation methods using microfluidic devices.In addition, techniques such as flow cytometers are specialized for separating and selecting biological particles such as cells. is also frequently used. However, existing methods have advantages and disadvantages in terms of ``separation accuracy,'' ``simplicity of operation,'' ``cost of separation equipment and systems,'' and ``separation speed,'' and there is still no technology that satisfies all of these. It is still in the process of development. Additionally, some techniques, such as normal filtration techniques, have the problem of ``objects smaller than a certain size can be collected, but objects larger than a certain size cannot be collected.'' Furthermore, when separating specific cells or biological particles for the purpose of diagnostic medicine, it is desirable that the separation device itself be disposable in order to prevent contamination between samples and ensure reproducibility. It is necessary that the device can be manufactured simply and efficiently. In addition, methods using microfluidic devices generally have the problem of being prone to clogging. Furthermore, it is also desirable that the method be capable of separating minute objects of about 100 nanometers to 1 micrometer. Below, we will provide an overview of the existing separation methods and their challenges.

Centrifugation and sedimentation are commonly used methods because they enable the separation of a relatively large amount of particles using simple methods. However, in order to perform highly accurate separation, a long and multi-step separation operation may be required. In particular, when separating biological particles such as cells for medical diagnostic purposes using conventional batch centrifugation, the disadvantage is that complicated and time-consuming separation operations are required by laboratory technicians with specialized skills. Another problem is that the accuracy of separation depends on the skill of the operator.

Filtration is a method that can realize a simple separation operation. As an example, JP2006-000848A shows a biopharmaceutical purification system that connects multi-stage separation units. Such systems and general syringe-mounted filter systems have the advantage of being easy to operate, disposable, and have a high throughput. However, it is difficult to recover large-sized components that have not passed through the filter, which is extremely disadvantageous in separating and purifying large-sized cells present in blood, for example.

JP2005-205387A shows an example of a method for separating particles using a microfluidic device. In this method, a particle suspension and a sheath liquid are introduced into a channel structure with branches, the particles are pressed against them in the narrow channel section, and then the width of the channel is increased to increase the direction of the force exerted on the particles by the flow. Particles are separated by size by taking advantage of the fact that they differ. This method allows precise separation with a simple method, and the device itself can be made disposable. It is also possible to separate the particles to be separated into large and small particles and collect them separately. However, there is a drawback in that the throughput is not sufficient, and when separating an object with a diameter of several micrometers, the throughput of the fine particle suspension is approximately several microliters per minute. Furthermore, it is necessary to use a narrow channel structure that is several times to ten times the diameter of the fine particles, which poses the problem of easy clogging.

JP6403190B describes a method for separating particles using a microchannel based on a different principle from JP2005-205387A. In this method, a main channel having an inclination and branch channels arranged in parallel to intersect with the main channel are combined to form a lattice-like channel structure, and fine particles are separated by size. In addition to the advantages of the method of JP2005-205387A, it has the effect of improving throughput and reducing the effects of clogging, but it still has the problem of not being able to separate submicrometer-sized particles, and Another problem is that the operation becomes complicated because it is necessary to introduce a solution that does not contain particles.

“Nature Protocols”, 2016 (11), 134-148. describes a method using a spiral microchannel that primarily targets cells. Since this technique uses a structure with a diameter of several hundred micrometers, clogging problems are less likely to occur and high separation throughput is achieved. Another advantage is that it can be separated by a simple operation using a disposable channel. However, like the methods shown in JP2005-205387A and JP6403190B, there is a problem that this method cannot be applied to separation of submicrometer-sized particles.

Furthermore, cross-flow filtration systems, which enable continuous filtration and separation, have also been widely used. JP2018-023926A shows an example of a cross-flow filtration system. In addition to this method, various cross-flow filtration systems have been developed, including separation devices for artificial dialysis. Cross-flow filtration systems generally allow for more precise separations, reduce the effects of clogging, and often achieve higher throughput than conventional filtration. However, there are many cases in which a disposable system is not assumed, and furthermore, in many cases, it is not assumed that biological particles such as cells are to be separated and purified and recovered. In particular, cross-flow filtration separation methods that are applicable to medical diagnosis and are intended for precise separation of cells and biological particles have hardly been put to practical use to date.

As mentioned above, various fine particle separation and purification devices have been proposed so far, but none of them have been able to "achieve a separation throughput of at least 0.1 mL per minute or more" with "simple operation" and "high accuracy". ``It is possible to collect objects larger than a certain size,'' ``It is less susceptible to clogging,'' ``It can be applied to objects as small as sub-micrometers,'' and ``It is possible to compare separation devices and systems.'' Very few particle separation/selection devices have been developed that can be easily produced at low cost and can be made into disposable systems.

The present invention has been made in view of the above-mentioned problems of the conventional technology, and its purpose is to achieve "with simple operation", "high accuracy", and "at least 0.000. It can achieve a separation throughput of about 1 mL or more," it can collect objects larger than a certain size, it is less susceptible to clogging, and it can be applied to objects from submicrometers to several tens of micrometers. The aim is to provide a novel separation device for biological particles. Furthermore, it is an attempt to provide a device that can be constructed in such a way that the device itself can be "produced relatively easily and at low cost" and can be "made into a disposable system."

In order to achieve the above object, the invention according to one aspect of the present invention is constructed by bonding a flat plate-like substrate A and a flat plate-shaped substrate B, respectively, and the substrate A has a structure in which communicating pores are formed inside. The region C is at least partially in contact with the bonding surface S of the substrate A and the substrate B, and the region C is at least partially in contact with the region C at the bonding surface S. An introduction channel E and a recovery channel F are formed, which are generally on the same plane, and the introduction channel E and the recovery channel F are arranged so as not to directly contact each other. The recovery channel F has at least one inlet I through which liquid can be introduced and at least one outlet J through which liquid can be discharged, and the recovery channel F has at least one outlet K through which liquid can be discharged, and is in contact with substrate B. This is a cross-flow filtration device that performs filtration in region C. By doing this, when a particulate suspension is continuously or instantaneously introduced from the inlet I, small particles that can pass through the porous region C are introduced from the introduction channel E to the recovery channel F. However, large particles that cannot pass through the region C pass through the introduction flow path E and are collected from the outlet J, making it possible to separate them with a simple operation. Furthermore, since the introduction channel E and the recovery channel F can be formed on the same plane, the device itself can be manufactured relatively easily and at low cost.

In addition, in the invention according to this aspect, although not limited to, the substrate A has a region D in which no communicating pores are formed inside and is at least partially in contact with the bonding surface S, It is desirable that the introduction flow path E includes a flow path portion G that is in contact with the joint surface S and that exists in a region D that exists between the region C and the outlet J. By doing this, flow resistance occurs in the flow path portion G connected to the outlet J, so that the liquid can be more efficiently led out to the recovery flow path F, and more efficient separation can be performed. can.

Further, in the invention according to this aspect, although not limited thereto, the introduction channel E and the recovery channel F may be arranged at least partially in a spiral shape parallel to each other. By doing this, the introduction channel E and the recovery channel F can be arranged at a relatively high density, and the device itself can be made compact, which not only reduces the cost of manufacturing the device, but also By controlling the interval between channel E and recovery channel F, it becomes possible to perform separation with higher precision.

Furthermore, in the invention according to this aspect, although not limited thereto, the introduction channel E may include at least two inlets I and I' through which liquid can be introduced. In this way, if a fine particle suspension is introduced continuously through the inlet I and a solution containing no fine particles is introduced continuously through the inlet I', separation with higher precision becomes possible.

Further, in the invention according to this aspect, although not limited to this, it is desirable that the diameter, width, or depth of the introduction channel E is at least partially 500 micrometers or less. By doing so, it becomes possible to target small particles of submicrometers to several tens of micrometers.

Furthermore, in the invention according to this aspect, it is desirable that the distance between the introduction channel E and the recovery channel F is 200 micrometers or more even at the shortest portion, although it is not limited thereto. By doing this, even with a short channel structure, it is possible to perform more efficient separation, improve throughput, and target small particles ranging from submicrometers to several tens of micrometers. This makes it possible to perform separation efficiently.

In addition, in the invention according to this aspect, although not limited to, region C includes mixing fine particles that can be dissolved in water into a rubber-like resin material before polymerization or in its molten state. The particles may be formed by polymerizing or solidifying the resin material and then dissolving the fine particles. By doing this, it is possible to reduce the manufacturing cost of the device itself, and by using, for example, sodium chloride particles as fine particles that can be dissolved in water, and by controlling the particle size and shape, more precise and It becomes possible to perform high-throughput and high-precision separation.

In addition, in the invention according to this aspect, although not limited to, region C is capable of dissolving a rubber-like resin material in an organic solvent that does not dissolve the resin material before polymerization or in its molten state. The resin material may be formed by mixing fine particles, polymerizing or solidifying the resin material, and then dissolving the fine particles. By doing this, it is possible to reduce the manufacturing cost of the device itself, and also to use particles made of polymethyl methacrylate (PMMA), polystyrene (PS), etc. as fine particles that can be dissolved in organic solvents. By controlling the diameter and shape, it becomes possible to perform separation with greater precision, higher throughput, and higher precision.

Since the present invention is configured as described above, cross-linking can be easily and reproducibly achieved by simply bonding a porous substrate and a non-porous substrate with a channel structure formed on either side. It becomes possible to create a flow filtration device. Therefore, it can be widely used as a disposable, general-purpose particle separation device.

Furthermore, since the present invention is configured as described above, it is possible to achieve a separation throughput of at least about 0.1 mL per minute with simple operation, with high precision, and at a constant It is possible to provide a new particulate separation device that can collect objects larger than the above size, is less susceptible to clogging, and is applicable to objects from submicrometers to several tens of micrometers in size. becomes.

FIG. 1 is a schematic diagram showing an example of a cross-flow filtration device according to an embodiment, and is a Z-arrow view of the top surface of a substrate A in FIG. 1c. FIG. 1 is a schematic diagram showing an example of a cross-flow filtration device according to an embodiment, and is a Z arrow view of the lower surface of a substrate B in FIG. 1c. It is a schematic diagram showing an example of a cross-flow filtration device according to an embodiment, and is a cross-sectional view taken along the line XX′ in FIGS. 1a and 1b of the cross-flow filtration device formed by joining substrate A and substrate B. be. 2b, 2c, and FIG. 2d are views showing a flow path structure common to FIG. 2d, and a Z arrow view in FIGS. 2b, 2c, and 2d. 1 is a schematic diagram showing an example of a cross-flow filtration device in which a flow path structure similar to the flow path structure in the cross-flow filtration device shown in FIGS. 1a to 1c is formed according to the embodiment, and the entire substrate A is a region 2a is a cross-sectional view taken along the line XX′ in FIG. 2a of a cross-flow filtration device configured by C and having a flow path structure formed on the lower surface of the substrate B. FIG. 1 is a schematic diagram showing an example of a cross-flow filtration device in which a flow path structure similar to the flow path structure in the cross-flow filtration device shown in FIGS. 2a is a cross-sectional view taken along the line XX' in FIG. 2a of a cross-flow filtration device in which a flow path structure is formed in a region C in which the cross-flow filtration device is formed. 1 is a schematic diagram showing an example of a cross-flow filtration device in which a flow path structure similar to the flow path structure in the cross-flow filtration device shown in FIGS. 1a to 1c is formed according to the embodiment, and the entire substrate A is a region 2a is a cross-sectional view taken along the line XX′ in FIG. 2a of a cross-flow filtration device configured by C and having a flow path structure formed on the lower surface of the substrate A. FIG. It is a schematic diagram schematically showing a channel structure formed on a joint surface S in a cross-flow filtration device according to an embodiment, and includes one linear introduction channel E and one linear recovery channel F. A cross-flow filtration device is shown. It is a schematic diagram schematically showing a channel structure formed on a joint surface S in a cross-flow filtration device according to an embodiment, and includes one linear introduction channel E and two linear recovery channels. 1 shows a cross-flow filtration device with F. It is a schematic diagram schematically showing a channel structure formed on a joint surface S in a cross-flow filtration device according to an embodiment, in which one introduction channel E having a channel portion G and two linear channels are shown. A cross-flow filtration device with a recovery channel F is shown. It is a schematic diagram showing a channel structure formed on the upper surface of a substrate A for a cross-flow filtration device according to an embodiment in which an introduction channel E and a recovery channel F are arranged in a spiral shape parallel to each other. The upper surface of the board|substrate A in the cross-flow filtration apparatus which has the flow path structure arrange|positioned in the spiral shape is shown. It is a schematic diagram showing the flow path structure formed on the lower surface of the substrate B regarding the cross flow filtration device according to the embodiment in which the introduction flow path E and the recovery flow path F are arranged in a spiral shape parallel to each other. It shows the lower surface of a substrate B in a cross-flow filtration device having a channel structure arranged in a spiral shape. It is a schematic diagram showing a channel structure formed on the upper surface of a substrate A for a cross-flow filtration device according to an embodiment in which an introduction channel E and a recovery channel F are arranged in a spiral shape parallel to each other. The upper surface of the board|substrate A in the cross-flow filtration apparatus which has the flow path structure arrange|positioned in the spiral shape is shown. It is a schematic diagram showing a channel structure formed on the lower surface of a substrate B in a cross-flow filtration device according to an embodiment in which an introduction channel E and a recovery channel F are arranged in a spiral shape parallel to each other. It shows the lower surface of a substrate B in a cross-flow filtration device having a channel structure arranged in a spiral shape. 1 is a schematic diagram of a cross-flow filtration device forming an introduction channel E with two inlets I and I′ according to an embodiment, showing the top surface of a substrate A; FIG. FIG. 2 is a schematic diagram of a cross-flow filtration device in which an introduction channel E with two inlets I and I' is formed according to an embodiment, and shows the lower surface of a substrate B in which an introduction channel E and a recovery channel F are formed. ing. 6C is a schematic view showing the form of a cross-flow filtration device used in Examples to separate fine particles having a diameter of several micrometers, and is a view taken along the Z arrow in FIG. 6C of the top surface of the substrate A. FIG. 6C is a schematic diagram showing the form of a cross-flow filtration device used in Examples to separate fine particles having a diameter of several micrometers, and is a view taken along the Z arrow in FIG. 6c of the lower surface of the substrate B. FIG. FIG. 6a is a schematic diagram showing the form of a cross-flow filtration device used to separate fine particles with a diameter of several micrometers in Examples, and FIG. 6a is a cross-flow filtration device formed by bonding substrate A and substrate B. and a sectional view taken along line XX' in FIG. 6b. FIG. 6 is a schematic diagram showing the form of a cross-flow filtration device used in Examples to separate fine particles having a diameter of several micrometers, and is an enlarged view of region d in FIG. 6b. FIG. 6 is a schematic diagram showing the form of a cross-flow filtration device used in Examples to separate fine particles having a diameter of several micrometers, and is an enlarged view of region e in FIG. 6b. This is a scanning electron microscope image of a porous region C formed using a silicone resin and sodium chloride fine particles in an example, and sodium chloride fine particles having a particle size of 30 to 60 micrometers and a particle size of 90 to 140 micrometers were used. This is a scanning electron microscope image of a cross section of region C formed by the method. This is a scanning electron microscope image of a porous region C formed using a silicone resin and sodium chloride fine particles in an example, and sodium chloride fine particles having a particle size of 30 to 60 micrometers and a particle size of 90 to 140 micrometers were used. This is a scanning electron microscope image of a cross section of region C formed by the method. This is a scanning electron microscope image of a porous region C formed using a silicone resin and sodium chloride fine particles in an example. This is a scanning electron microscope image. 1 is a scanning electron microscope image of a porous region C formed using a silicone resin and polymethyl methacrylate fine particles having an average diameter of about 5 micrometers in an example, and a scanning electron microscope image of a cross section of the region C. 1 is a scanning electron microscope image of a porous region C formed using a silicone resin and polymethyl methacrylate fine particles having an average diameter of about 5 micrometers in an example, and a scanning electron microscope image of the surface of region C. FIG. 9d is a schematic diagram showing another form of a cross-flow filtration device consisting of three substrates, which was used to separate fine particles with a diameter of several micrometers in the examples, and FIG. 9d shows the bottom surface of the topmost substrate. FIG. FIG. 9 is a schematic diagram showing another form of a cross-flow filtration device consisting of three substrates, which was used to separate fine particles with a diameter of several micrometers in Examples, and shown in the Z arrow view in FIG. 9d on the bottom surface of substrate A. It is a diagram. FIG. 9 is a schematic diagram showing another form of a cross-flow filtration device consisting of three substrates, which was used to separate fine particles with a diameter of several micrometers in Examples, and shown in the Z arrow view in FIG. 9d on the top surface of substrate B. It is a diagram. FIG. 2 is a schematic diagram showing another form of a cross-flow filtration device consisting of three substrates, which was used to separate fine particles with a diameter of several micrometers in the examples, in which the uppermost substrate, substrate A, 9a, 9b, and 9c of a cross-flow filtration device formed by bonding substrate B and substrate B together. FIG. This is a graph showing the particle separation behavior using the cross-flow filtration device shown in FIG. On the other hand, with outlet J-2 and outlet K-2 blocked, a suspension containing standard polystyrene fine particles with an average diameter of 0.29 to 3.2 micrometers was introduced from inlet I, and outlet J-1 and outlet 1 is a graph showing the number ratio of each fine particle contained in the solution recovered from K-1.

EMBODIMENT OF THE INVENTION Hereinafter, the best mode regarding the cross flow filtration apparatus based on this invention shall be demonstrated in detail. However, the present invention can be implemented in many different forms and is not limited to the embodiments and examples shown below.

1a to 1c are schematic diagrams showing an example of a cross-flow filtration device, in which FIG. 1a is a view of the top surface of substrate A in the direction of the Z arrow in FIG. 1c, and FIG. 1b is a view of the bottom surface of substrate B. 1c is a Z arrow view in FIG. 1c, and FIG. 1c is a cross-sectional view taken along line XX' in FIGS. 1a and 1b of a cross-flow filtration device formed by joining substrate A and substrate B.

In the cross-flow filtration apparatus shown in FIGS. 1a to 1c, a substrate A is composed of a porous region C having communicating holes and a non-porous region D. Further, the region C is formed to be exposed on the upper surface of the substrate A, and this surface corresponds to the bonding surface S.

The substrates A and B shown in FIGS. 1a to 1c can be physically fixed using a jig or adhesive tape, chemically directly bonded using oxygen plasma, UV ozone, a silane coupling agent, etc., or bonded by adhesive coating. Bonding can be performed by using techniques such as bonding, thermocompression bonding, anodic bonding, or any combination thereof.

The introduction channel E and the recovery channel F have rectangular cross sections with uniform width. However, the shape other than the rectangle may be a flow path structure having various cross-sectional shapes such as a polygon other than a quadrangle, a trapezoid, a semicircle, a circle, etc. However, a channel structure having a rectangular shape is preferable in the sense that it is easy to manufacture and it is easy to precisely control the intervals between channels.

In the cross-flow filtration apparatus shown in FIGS. 1a to 1c, two recovery channels F are formed at equal intervals so as to sandwich the introduction channel E, and each has an outlet K, so there are two recovery channels F in total. Exit K exists.

The introduction channel E is arranged so as to be partially in contact with a region C having communicating pores, and the channel portion G in the introduction channel E is arranged so as to be in contact with a non-porous region D. ing.

The diameter, width, or depth of the introduction channel E may be of any value as long as it can separate fine particles with a diameter of several micrometers or smaller. However, from the viewpoint of performing precise separation, these values are preferably at least partially 500 micrometers or less, and particularly 200 micrometers or less in the case of a channel structure with a rectangular cross section. is more preferable.

From the viewpoint of separating the fine particles contained in the suspension introduced from the inlet I using the communicating holes present in the region C, the introduction channel E and the recovery channel F are independent from each other and exist without contact. It is necessary to do so. In this case, the introduction flow path E and the recovery flow path F are indirectly connected through the communication hole present in the region C.

The interval between the introduction channel E and the recovery channel F can take any value as long as it is possible to separate the particles to be separated. However, from the viewpoint of keeping the size distribution of the communicating pores formed in region C constant and achieving hydraulic separation, the value is preferably 200 micrometers or more even at its smallest.

The introduction channel E and the recovery channel F may be configured by a microchannel structure fabricated using microfabrication technology. By doing so, it is possible to use a channel structure whose size and shape are arbitrarily and accurately controlled, and therefore it is possible to arbitrarily control the behavior and efficiency of particle separation. In addition, such microfabrication techniques include injection molding, replica molding, direct machining by cutting, laser processing, electron beam direct writing, three-dimensional modeling, three-dimensional stereolithography, wet etching, dry etching, embossing, imprinting, Various methods such as can be used.

When producing the introduction channel E and the recovery channel F using microfabrication technology, various materials can be used as the base material constituting the substrate B shown in FIGS. 1a to 1c. As examples, silicone resins such as PDMS (polydimethylsiloxane), thermoplastic elastomers, various polymer materials such as acrylic, ceramic materials including glass, and various metal materials such as stainless steel can be used. It is also possible to use a combination of arbitrary plural types of substrates among the materials. Furthermore, in order to prevent fine particle adsorption onto the channel surface, it is also possible to surface-modify these base materials with polyethylene glycol, a polymer having a phospholipid polar group, polymethoxyethyl acrylate, etc. It is also possible to use a resin base material in which these polymers are mixed, a base material in which a resin in which these polymers are chemically bonded, and the like are mixed. Note that in order to provide a disposable cross-flow filtration system, it is preferable to use a relatively inexpensive polymer material.

In the cross-flow filtration apparatus shown in FIGS. 1a to 1c, the substrate B may be formed of a porous material. However, even if pores are formed in the substrate B, the effect thereof is limited, so the pores do not necessarily need to be formed of a porous material.

As the material for the substrate A, a wide variety of materials similar to those for the substrate B can be used. Note that the region C and the region D may be made of different materials, or may be made of the same material. Furthermore, the materials constituting the substrate A and the substrate B may be the same, partially different, or entirely different.

The region C may have any shape and size as long as the introduction channel E and the recovery channel F can be brought into contact with each other efficiently. However, from the viewpoint of joining flat plates, it is preferable that at least a portion thereof be configured in a planar manner. Furthermore, by making the region C surrounded by the region D except for the part that is in contact with the bonding surface S, the solution introduced from the inlet I can be made to flow out only from the outlet K and the outlet J. It is preferable to do so.

In the production of porous region C, dissolvable fine particles are mixed with a rubber-like resin material before its polymerization or in its molten state, and after the resin material is polymerized or solidified, the fine particles are dissolved. It is also possible to use a method. By doing so, the matrix of region C having communicating pores can be produced inexpensively, easily, and with good reproducibility. As the rubber-like resin material, silicone resin and thermoplastic elastomer are suitable. Further, the volume ratio of the fine particles mixed in the resin material may be any value as long as communicating pores can be formed, but it is generally preferably in the range of 30 to 60%.

In particular, when substrate A and substrate B are made of silicone resin, there is an advantage that they can be easily and firmly bonded by chemically activating them with oxygen plasma, etc. It is preferable to use silicone resin as the material.

In order to form communicating pores in region C, various fine particles that can be dissolved in water or an organic solvent can be used. For example, any water-soluble particles may be used, such as sodium chloride particles, potassium chloride particles, phosphate particles, sucrose particles, glucose particles, etc. Furthermore, various fine particles such as polymethyl methacrylate particles and polystyrene particles can be used as the fine particles that can be dissolved in an organic solvent. Furthermore, by controlling the diameters of these fine particles, the separation behavior of the fine particles to be separated can be controlled. The size of the particles to be dissolved is expected to be in the range of about 0.1 micrometer to 1 millimeter in terms of average diameter, but when separating objects from submicrometers to several micrometers, It is preferable to select fine particles with a size of about 10 micrometers to 500 micrometers. By using such fine particles, it is possible to form communication holes having a size corresponding to the diameter of the fine particles. Note that when using fine particles that can be dissolved in an organic solvent, it is preferable to select an organic solvent that does not dissolve region C, or to form region C using a material that does not dissolve in the organic solvent.

When the region C is formed in a planar manner, its thickness may have any value as long as the purpose of separation is achieved. However, if the thickness is thinner than the diameter of the fine particles to be dissolved, it will be difficult to form communicating holes, so it is preferable to have a thickness of about three times the diameter of the fine particles to be dissolved. Generally, it is preferably in the range of about 0.1 to 5 mm.

2a to 2d are schematic diagrams illustrating examples of three types of cross-flow filtration devices that have flow path structures similar to those in the cross-flow filtration devices shown in FIGS. 1a to 1c. FIG. 2a is a diagram showing a flow path structure common to FIGS. 2b, 2c, and 2d, and is a view taken along the Z arrow in FIGS. 2b, 2c, and 2d. FIG. 2b shows a cross-flow filtration device in which the entire substrate A is constituted by a region C and a flow channel structure is formed on the lower surface of the substrate B. FIG. 2d shows a cross-flow filtration device in which the entire substrate A is constituted by region C and a flow path structure is formed on the lower surface of the substrate A, respectively, XX in FIG. 2a. FIG.

As shown in FIGS. 2a to 2d, various patterns can be assumed as the positional relationship between the substrate A including the region C, the substrate B, and the channel structure. The introduction channel E and the recovery channel F may be formed on the substrate A side, may be formed on the substrate B side, or may be formed on both substrate A and substrate B. Further, the substrate A may not have the region D and may be composed of the region C, or at least a portion of the substrate B may be formed of a porous material.

3a to 3c are schematic diagrams schematically showing the channel structures formed on the joint surface S in three types of cross-flow filtration devices, and FIG. 3a is a straight introduction channel E. 3b is a cross-flow filtration device having one straight introduction channel E and two straight recovery channels F, FIG. 3c shows a cross-flow filtration device having one introduction channel E having a channel portion G and two straight recovery channels F.

As shown in FIGS. 3a to 3c, various patterns can be assumed for the structure of the introduction channel E and the recovery channel F. In this way, as long as there is at least one inlet I and one outlet J in the introduction channel E, and at least one outlet K in the recovery channel F, there can be a plurality of introduction channels E and recovery channels F. It doesn't matter if it exists. In addition, when there are recovery channels F on both sides of the introduction channel E, these channels do not necessarily have to be arranged at equal intervals, but the recovery channels are located at equal positions on both sides of the introduction channel E. The presence of F is preferable from the viewpoint of easy control of separation accuracy.

The flow path portion G may or may not be formed. However, the presence of flow path portion G increases the resistance to flow to outlet J, making it possible to increase the relative flow rate to outlet K, allowing more efficient separation. . Therefore, it may be preferable that the channel portion G is formed. Note that by appropriately adjusting the length, width, and depth of the channel portion, it is possible to control the behavior of particle separation.

FIGS. 4a to 4d show two types of cross-flow filtration devices in which an introduction channel E and a recovery channel F are arranged in a spiral shape parallel to each other. Schematic diagrams showing channel structures are shown, with FIG. 4a showing the top surface of a substrate A in a cross-flow filtration device having a channel structure arranged in a circular spiral shape, and FIG. 4b showing a channel structure arranged in a circular spiral shape. FIG. 4c shows the bottom surface of substrate B in a cross-flow filtration device having a channel structure arranged in a rectangular spiral shape, and FIG. 4d shows the lower surface of the substrate B in a cross-flow filtration device having a channel structure arranged in a rectangular spiral shape.

As shown in FIGS. 4a to 4d, by arranging the introduction channel E and the recovery channel F alternately so as to spread outward from the center, it is possible to arrange only one channel each. , efficient separation becomes possible. In addition, by doing so, the channel structure can be arranged in a compact and dense manner in a limited space, so that the device itself can be miniaturized, which is preferable.

Note that, as shown in FIGS. 4a to 4d, the term "helical shape" in this case can take various forms such as not only a circle but also a rectangle and a polygon. However, by arranging the channel structure in a circular or rectangular spiral shape, the channel can be designed more smoothly.

Further, unlike the channel structure shown in FIGS. 4a to 4d, a spiral arrangement may be used in which the inlet I is located on the outside and the exits J and K are located on the center side.

When a suspension of fine particles is continuously introduced from inlet I into the channel structure shown in Figures 1a to 4d, these fine particles are continuously separated due to differences in size or deformability, and are separated into separate particles. Collected from the exit.

Various means can be used to continuously introduce a suspension of microparticles. The simplest method is to connect a syringe or the like to inlet I and pressurize it manually or with a pump to continuously introduce a suspension of microparticles. You can also use the system. Furthermore, by suctioning the solution from the outlet J and the outlet K, a suspension of fine particles may be continuously introduced into the flow path.

Possible particles to be separated include various synthetic particles, cultured cells, cells or biological particles existing in blood, environmental particles, liposomes, and vesicles. It is preferable to use a solution that can stably disperse these fine particles, and in particular, when separating cells or biological particles, it is preferable to use an aqueous solution with a buffering effect.

When a solution in which these separation targets are suspended is introduced from inlet I, particles smaller than a certain size or particles with high deformability pass through porous region C and are introduced into recovery channel F. , and is finally discharged from outlet K. On the other hand, particles larger than a certain size or particles with low deformability flow downstream through the introduction channel E and are finally discharged from the outlet J. At this time, a certain proportion of particles smaller than a certain size are also discharged from the outlet J at the same time as larger particles.

In addition, the size of the pores formed in the region C is small, the distance between the introduction channel E and the recovery channel F is long, the depth value of the introduction channel E is small, and the flow rate distributed to the outlet J. The higher the ratio, the smaller the cutoff size for separation, that is, it becomes possible to separate only smaller particles.

5a and 5b show a schematic diagram of a cross-flow filtration device forming an inlet channel E with two inlets I and I', FIG. 5a showing the top side of the substrate A, FIG. 5b shows the bottom surface of the substrate B on which the introduction channel E and the recovery channel F are formed.

The introduction channel E shown in FIGS. 5a and 5b has only one inlet by continuously introducing a particle suspension from the inlet I and a solution containing no particles from the inlet I'. Compared to the introduction channel E, more accurate separation is achieved. High precision here means that the proportion of small particles mixed into the solution recovered from the outlet J is reduced.

The effects of the present invention were confirmed by actually producing a cross-flow filtration system according to the above embodiment and performing a particle separation experiment. This will be explained below.

6a to 6e are schematic diagrams showing the configuration of a cross-flow filtration device used to separate fine particles with a diameter of several micrometers, and FIG. 6a is a Z-arrow view of the top surface of substrate A in FIG. 6b is a Z-arrow view of the lower surface of substrate B in FIG. 6c, and FIG. 6c is a cross-flow filtration device formed by joining substrates A and B, taken along line XX' in FIGS. 6a and 6b. FIG. 6d and FIG. 6e are enlarged views of region d and region e in FIG. 6b, respectively.

The flow channel structure on the bottom surface of the substrate B shown in FIGS. 6a to 6e was created by fabricating a mold made of negative photoresist by soft lithography, and applying polydimethylsiloxane (PDMS) prepolymer, a type of silicone resin, to the mold. It was formed by casting and polymerizing. Inlet I, outlet J, and outlet K are formed by punching holes. Note that the thickness of the substrate B was about 3 mm.

Substrate A shown in Figures 6a to 6e is made by mixing PDMS with sodium chloride fine particles at a volume ratio of 50%, casting it into a flat plate, polymerizing it, and then casting PDMS that does not contain sodium chloride fine particles around it. After that, the formed base material was immersed in distilled water to dissolve the sodium chloride fine particles and form micropores communicating with the portion corresponding to region C. As the sodium chloride fine particles, those having particle diameters in the range of 30 to 60 micrometers and those in the range of 90 to 140 micrometers were used. The thickness of region C was 1 mm, and the thickness of the entire substrate A was about 2 mm.

The cross-flow filtration system shown in FIGS. 6a to 6e was formed by activating substrate A and substrate B by oxygen plasma treatment and bonding them together.

In the channel structure shown in FIGS. 6a to 6e, both the introduction channel E and the recovery channel F have a depth of about 60 micrometers, and the introduction channel E and the introduction channel other than the channel section G have a depth of about 60 micrometers. The widths of channel portion G and recovery channel F in channel E were 100 micrometers, 50 micrometers, and 200 micrometers, respectively. Further, the introduction channel E and the recovery channel F were arranged in a rectangular spiral shape, and the distance between these channels was 350 micrometers. Further, the length of the introduction channel E other than the channel section G was 10 cm, and the length of the channel section G was 10 or 30 cm. The flow path portion G was also arranged so that it could be folded compactly into a spiral shape.

Figures 7a to 7c show scanning electron microscopy images of porous region C formed using PDMS and sodium chloride microparticles, with particle sizes of 30-60 micrometers in Figures 7a and 7b, respectively. FIG. 7c is a scanning electron microscope image of a cross section of region C formed using sodium chloride fine particles with a particle size of 90 to 140 micrometers, and FIG. This is a scanning electron microscope image of the surface of .

As shown in FIGS. 7a to 7c, when these two types of sodium chloride fine particles with different sizes are used, pores are formed by dissolving the sodium chloride fine particles, and the pores partially contact each other, Formation of communicating holes was observed. Furthermore, it was observed that a large number of micropores were formed on the surface of the porous region C.

Figures 8a and 8b show scanning electron microscopy images of porous region C formed using silicone resin and polymethyl methacrylate microparticles with an average diameter of about 5 micrometers; FIG. 8b is a scanning electron microscope image of a cross section of region C, and FIG. 8b is a scanning electron microscope image of the surface of region C.

As shown in FIGS. 8a and 8b, pores are formed by removing and dissolving the polymethyl methacrylate fine particles using an organic solvent, and the pores also partially contact each other to form communicating pores. The situation was observed. Furthermore, it was observed that a large number of micropores were formed on the surface of the porous region C.

Figures 9a to 9d show schematic diagrams of other configurations of a three-substrate cross-flow filtration device used to separate particles with a diameter of several micrometers; 9d is a view of the lower surface of the uppermost substrate in the direction of the Z arrow in FIG. 9d, FIG. 9b is a view of the lower surface of the substrate A in the direction of the Z arrow in FIG. 9d, and FIG. 9d is a cross-sectional view taken along line XX' in FIGS. 9a, 9b, and 9c of a cross-flow filtration device formed by joining the uppermost substrate, substrate A, and substrate B.

The cross-flow filtration devices shown in FIGS. 9a-9d were constructed similarly to the cross-flow filtration devices shown in FIGS. 6a-6e. The channel structure in the cross-flow filtration device shown in FIGS. 9a to 9d is formed by bonding three substrates, and the introduction channel E existing on the bottom surface of the substrate A is divided into eight in the middle. The branches are finally joined at the lower surface of the substrate located at the top, and connected to the exit J-1 or the exit J-2. In addition, recovery channels are formed on both sides of each of the eight branched introduction channels on the bottom surface of the substrate A, and these channels merge on the bottom surface of the substrate A, pass through the channel section G, and exit K- 1 or K-2.

In this channel, two outlets J and two outlets K are provided, and by using either one of these, the flow rate at the outlet can be adjusted and the separation size can be controlled. Be expected.

The width of the introduction channel branched on the lower surface of substrate A was 100 micrometers, the width of the recovery channel was 200 micrometers, and the depths of both were about 50 micrometers.

For the cross-flow filtration apparatus shown in FIGS. 9a to 9d, standard fluorescent microparticles of polystyrene are suspended in an aqueous solution containing 18% sucrose and 0.5% Tween 20, and a syringe pump is used to remove the suspension. was continuously introduced through inlet I at a flow rate of 100 microliters per minute.

FIG. 10 is a graph showing the particle separation behavior using the cross-flow filtration apparatus shown in FIGS. 9a to 9d. With outlet J-2 and outlet K-2 blocked, a suspension containing standard polystyrene fine particles with an average diameter of 0.29 to 3.2 micrometers is introduced into the filtration device from inlet I, and outlet J- 1 and a graph showing the number ratio of each fine particle contained in the solution collected from outlet K-1. This graph calculates the number of particles collected from each outlet by multiplying the particle concentration in the collected liquid by the volume of the liquid, and displays the total number of particles collected from the two outlets as 100%. It is something that

As shown in Figure 10, when using a cross-flow filtration device with region C formed using sodium chloride fine particles with a diameter of 30-60 micrometers, most of the 3.2 micrometer particles are collected from outlet J. It was done. At this time, the amount of solution flowing out from outlet K was about 50 to 60% of the total amount. The smaller the introduced particle size, the more the proportion of particles introduced into the collection channel and recovered from outlet K-1 increases; in the case of particles of 0.29 micrometers, there are two outlets approximately according to the flow rate distribution. It was confirmed that it was recovered. In other words, this device is applicable to the separation of microparticles from micrometer to submicrometer size.

It should be noted that most of the 3.2 micrometer fine particles were collected from outlet J-1. The size of the pores formed on the surface of region C was about 5 micrometers, but since these 3.2 micrometer particles did not pass through the porous part, the cross-flow filtration device of the present invention It was suggested that not only the physical pore diameter of the communicating pores but also hydraulic effects including flow distribution determined the size of the separated particles. This suggests that the cross-flow filtration device of the present invention is a separation device that is less susceptible to clogging.

Since the present invention is configured as described above, "by the simple operation of introducing a fine particle suspension," "with high precision," and "at least 0.1 mL or more of separation per minute can be achieved." A continuous separation device that can achieve throughput, can collect objects larger than a certain size, is less susceptible to clogging, and can be applied to objects as small as sub-micrometers. It is extremely useful as a Therefore, it has various industrial applications, such as the preparation of various synthetic fine particles, the removal of fine particulate impurities in bi-processes such as antibody production, the separation of various cells, and the pretreatment of body fluid samples such as blood for disease diagnosis. Be expected.

Furthermore, since the present invention is configured as described above, it exhibits excellent effects such as ``separation devices and systems can be produced relatively easily and at low cost'' and ``the system can be made disposable.'' . Therefore, as an inexpensive and versatile disposable filter device that can be used with ease of use similar to a syringe filter, it can be expected to be used as a new device useful in various research and development fields such as biochemical research and environmental analysis.

Claims (5)

It is constructed by joining flat plate-shaped substrates A and B, respectively,
The substrate A has a region C in which communicating pores are formed,
Region C is at least partially in contact with bonding surface S of substrate A and substrate B,
An introduction channel E and a recovery channel F are formed, each of which is at least partially in contact with the region C at the joint surface S and is at least partially coplanar;
The introduction channel E and the recovery channel F are arranged so as not to be in direct contact with each other,
The introduction channel E has at least one inlet I through which liquid can be introduced and at least one outlet J through which liquid can be discharged,
The recovery channel F has at least one outlet K through which liquid can be discharged,
The substrate A has a region D in which no communicating pores are formed inside and is at least partially in contact with the bonding surface S,
The introduction flow path E includes a flow path portion G that is in contact with the joint surface S and in contact with the region D between the region C and the outlet J,
filtering in a region C in contact with the substrate B;
Crossflow filtration equipment. The introduction channel E and the recovery channel F are at least partially arranged in a spiral shape parallel to each other,
The cross-flow filtration device according to claim 1 . The introduction channel E is equipped with at least two inlets I and I' through which liquid can be introduced.
The cross-flow filtration device according to any one of claims 1 to 2 . The diameter, width or depth value of the introduction channel E is at least partially less than or equal to 500 micrometers;
The cross-flow filtration device according to any one of claims 1 to 3 . The distance between the introduction channel E and the recovery channel F is 200 micrometers or more even at the shortest part.
The cross-flow filtration device according to any one of claims 1 to 4 .